In article <username-190295163021 at slip7-5.acs.ohio-state.edu> username at osu.edu (Your Name) writes:
>I have recently encountered the technique of Systematic Evolution of
>Ligands by EXponential enrichment whereby a random RNA population generated
>via in vitro T7 transcription of synthetic DNA templates is "enriched" (by
>chromatography?) through a column containing the ligand (transition state
>analog for ribozyme selection, I would assume) of which one wants the RNA
>to bind. Those RNAs that are retained in the column are then cloned and
>analyzed for activity. Now then, I am wondering if anyone knows of a
>specific example of a catalytic RNA synthesized in this fashion and the
>reaction catalyzed, including all appropriate constants etc... I am
>familiar with Thom Cech's work and others, but this was heretofore unknown
>by myself.
The technique was simultaneously invented by at least three groups;
Larry Gold's, Jack Szostak's, and Gerry Joyce's. The technique was
dubbed SELEX by Tuerk and Gold, and the selected RNA molecules were
dubbed "aptamers" by Ellington and Szostak. The enrichment step can be
done in a variety of ways, including column affinity chromatography
and nitrocellulose filter binding. An amplification step by PCR, as
part of an iterative selection/amplification, is an important part of
the protocol -- it is not coincidence that SELEX followed shortly on
the heels of PCR. Techniques based on enrichment alone had previously
been used for studying the targets of some DNA-binding proteins.
SELEX has been used most often to generate ligands, rather than
enzymes, because this is easier. Two papers in which SELEX was used to
generate a catalyst are:
@Article{Robertson90,
author = "D.L. Robertson and G.F. Joyce",
title = "Selection in vitro of an {RNA} Enzyme that
Specifically Cleaves Single-Stranded {DNA}",
journal = "Nature",
year = 1990,
volume = 344,
pages = "467-468"
}
@Article{Bartel93,
author = "David P. Bartel and Jack W. Szostak",
title = "Isolation of New Ribozymes from a Large Pool of
Random Sequences",
journal = "Science",
year = 1993,
volume = 261,
pages = "1411-1418"
}
--
- Sean Eddy
- MRC Laboratory of Molecular Biology, Cambridge, England
- sre at mrc-lmb.cam.ac.uk